Peripheral blood B-cell death compensates for excessive proliferation in lymphoid tissues and maintains homeostasis in bovine leukemia virus-infected sheep.
; Gillet, Nicolas ; et al
in Journal of Virology (2006), 80(19), 9710-9719
The size of a lymphocyte population is primarily determined by a dynamic equilibrium between cell proliferation and death. Hence, lymphocyte recirculation between the peripheral blood and lymphoid tissues ... [more ▼]
The size of a lymphocyte population is primarily determined by a dynamic equilibrium between cell proliferation and death. Hence, lymphocyte recirculation between the peripheral blood and lymphoid tissues is a key determinant in the maintenance of cell homeostasis. Insights into these mechanisms can be gathered from large-animal models, where lymphatic cannulation from individual lymph nodes is possible. In this study, we assessed in vivo lymphocyte trafficking in bovine leukemia virus (BLV)-infected sheep. With a carboxyfluorescein diacetate succinimidyl ester labeling technique, we demonstrate that the dynamics of lymphocyte recirculation is unaltered but that accelerated proliferation in the lymphoid tissues is compensated for by increased death in the peripheral blood cell population. Lymphocyte homeostasis is thus maintained by biphasic kinetics in two distinct tissues, emphasizing a very dynamic process during BLV infection. [less ▲]Detailed reference viewed: 72 (12 ULg)
Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep.
Florins, Arnaud-Francois ; Gillet, Nicolas ; et al
in Journal of virology (2006), 80(24), 11998-2008
Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown ... [more ▼]
Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics. [less ▲]Detailed reference viewed: 39 (17 ULg)
Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester (CFSE).
; ; Florins, Arnaud-Francois et al
in Proceedings of the Royal Society B : Biological Sciences (2006), 273(1590), 1165-71
The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells ... [more ▼]
The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells irrespective of their stage in the cell cycle makes it preferable to deuterated glucose and BrdU, which only label dividing cells and thus produce unrepresentative results. In the past, experiments have been limited by the need to obtain a clear separation of CFSE peaks forcing scientists to adopt a strategy of in vitro labelling of cells followed by their injection into the host. Here we develop a framework for analysis of in vivo CFSE labelling data. This enables us to estimate the rate of proliferation and death of lymphocytes in situ, and thus represents a considerable advance over current procedures. We illustrate this approach using in vivo CFSE labelling of B lymphocytes in sheep. [less ▲]Detailed reference viewed: 57 (20 ULg)
The homeobox protein MSX2 interacts with tax oncoproteins and represses their transactivation activity.
Twizere, Jean-Claude ; ; et al
in Journal of Biological Chemistry (2005), 280(33), 29804-11
Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to ... [more ▼]
Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to immortalize primary cells in vitro. To gain insight into the molecular pathways mediating the activities of this important gene, we identified cellular proteins interacting with Tax. By means of a two-hybrid approach, we show that Tax specifically interacts with MSX2, a general repressor of gene expression. GST pull-down experiments and co-immunoprecipitation assays further confirmed binding specificity. Furthermore, the N-terminal residues 1-79 of MSX2 are required for binding, whereas the C-terminal residues 201-267 of MSX2 do not play a critical role. Whereas the oncogenic potential of Tax in primary cells was only slightly affected by overexpression of MSX2, the other function of Tax, namely LTR-dependent transcriptional activation, was inhibited by MSX2 in human HeLa and bovine B-lymphoblastoid (BL3) cell lines. This MSX2 repression function can be counteracted by overexpression of transcription factors CREB2 and RAP74. The Tax/MSX2 interplay thus results in repression of viral transcriptional activation possibly acting as a regulatory feedback loop. Importantly, this viral gene silencing is not strictly associated with a concomitant loss of Tax oncogenicity as measured by its ability to immortalize primary cells. And interestingly, MSX2 also interacts with and inhibits the transactivation function of the related Tax1 protein encoded by the Human T-cell leukemia virus type 1 (HTLV-1). [less ▲]Detailed reference viewed: 61 (25 ULg)
Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo.
; Florins, Arnaud-Francois ; Gillet, Nicolas et al
in Proceedings of the National Academy of Sciences of the United States of America (2005), 102(29), 10309-14
Leukemogenic viruses like human T-lymphotropic virus and bovine leukemia virus (BLV) presumably persist in the host partly by latent integration of the provirus in a fraction of infected cells, leading to ... [more ▼]
Leukemogenic viruses like human T-lymphotropic virus and bovine leukemia virus (BLV) presumably persist in the host partly by latent integration of the provirus in a fraction of infected cells, leading to accumulative increase in the outgrowth of transformed cells. Furthermore, viral infection also correlates with a blockade of the apoptotic mechanisms concomitant with an apparent latency of the host cell. Conceptually, induction of viral or cellular gene expression could thus also be used as a therapeutic strategy against retroviral-associated leukemia. Here, we provide evidence that valproate, an inhibitor of deacetylases, activates BLV gene expression in transient transfection experiments and in short-term cultures of primary B-lymphocytes. In vivo, valproate injection into newly BLV-inoculated sheep did not abrogate primary infection. However, valproate treatment, in the absence of any other cytotoxic drug, was efficient for leukemia/lymphoma therapy in the sheep model leading to decreased lymphocyte numbers (respectively from 25.6, 35.7, and 46.5 x 10(3) cells per mm3 to 1.0, 10.6, and 24.3 x 10(3) cells per mm3 in three leukemic sheep) and tumor regression (from >700 cm3 to undetectable). The concept of a therapy that targets the expression of viral and cellular genes might be a promising treatment of adult T cell leukemia or tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy, diseases for which no satisfactory treatment exists so far. [less ▲]Detailed reference viewed: 81 (31 ULg)
Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1.
; ; et al
in Oncogene (2005), 24(51), 7514-23
Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is ... [more ▼]
Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV. [less ▲]Detailed reference viewed: 18 (9 ULg)
Fate of premalignant clones during the asymptomatic phase preceding lymphoid malignancy.
; ; et al
in Cancer Research (2005), 65(4), 1234-43
Almost all cancers are preceded by a prolonged period of clinical latency during which a combination of cellular events helps move carcinogen-exposed cells towards a malignant phenotype. Hitherto ... [more ▼]
Almost all cancers are preceded by a prolonged period of clinical latency during which a combination of cellular events helps move carcinogen-exposed cells towards a malignant phenotype. Hitherto, investigating the fate of premalignant cells in vivo remained strongly hampered by the fact that these cells are usually indistinguishable from their normal counterparts. Here, for the first time, we have designed a strategy able to reconstitute the replicative history of the bona fide premalignant clone in an animal model, the sheep experimentally infected with the lymphotropic bovine leukemia virus. We have shown that premalignant clones are early and clearly distinguished from other virus-exposed cells on the basis of their degree of clonal expansion and genetic instability. Detectable as early as 0.5 month after the beginning of virus exposure, premalignant cells displayed a two-step pattern of extensive clonal expansion together with a mutation load approximately 6 times higher than that of other virus-exposed cells that remained untransformed during the life span of investigated animals. There was no fixation of somatic mutations over time, suggesting that they regularly lead to cellular death, partly contributing to maintain a normal lymphocyte count during the prolonged premalignant stage. This equilibrium was finally broken after a period of 18.5 to 60 months of clinical latency, when a dramatic decrease in the genetic instability of premalignant cells coincided with a rapid increase in lymphocyte count and lymphoma onset. [less ▲]Detailed reference viewed: 13 (9 ULg)
Involvement of glutathione as a mechanism of indirect protection against spontaneous ex vivo apoptosis associated with bovine leukemia virus.
; ; et al
in Journal of virology (2004), 78(12),Detailed reference viewed: 19 (7 ULg)
Le BLV: un modele d'etude pour les leucemies humaines.
in Bulletin et Mémoires de l'Académie Royale de Médecine de Belgique (2004), 159(10-12),Detailed reference viewed: 14 (3 ULg)
Investigation of the susceptibility of human cell lines to bovine herpesvirus 4 infection: Demonstration that human cells can support a nonpermissive persistent infection which protects them against tumor necrosis factor alpha-induced apoptosis
Gillet, Laurent ; ; et al
in Journal of Virology (2004), 78(5), 2336-2347
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we ... [more ▼]
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii) BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered. [less ▲]Detailed reference viewed: 71 (15 ULg)
Suppression Of Tumor Growth And Cell Proliferation By P13(Ii), A Mitochondrial Protein Of Human T Cell Leukemia Virus Type 1
; ; et al
in Proceedings of the National Academy of Sciences of the United States of America (2004), 101(17),Detailed reference viewed: 11 (3 ULg)
Reduced proviral loads during primo-infection of sheep by Bovine Leukemia virus attenuated mutants.
; ; et al
in Retrovirology (2004), 1(1),Detailed reference viewed: 13 (2 ULg)
Overlapping Cre And E Box Motifs In The Enhancer Sequences Of The Bovine Leukemia Virus 5 ' Long Terminal Repeat Are Critical For Basal And Acetylation-Dependent Transcriptional Activity Of The Viral Promoter: Implications For Viral Latency
; ; et al
in Journal of Virology (2004), 78(24),Detailed reference viewed: 38 (10 ULg)
Interaction of retroviral Tax oncoproteins with tristetraprolin and regulation of tumor necrosis factor-alpha expression.
Twizere, Jean-Claude ; ; et al
in Journal of the National Cancer Institute (2003), 95(24), 1846-59
BACKGROUND: The Tax oncoproteins are transcriptional regulators of viral expression involved in pathogenesis induced by complex leukemogenic retroviruses (or delta-retroviruses, i.e., primate T-cell ... [more ▼]
BACKGROUND: The Tax oncoproteins are transcriptional regulators of viral expression involved in pathogenesis induced by complex leukemogenic retroviruses (or delta-retroviruses, i.e., primate T-cell leukemia viruses and bovine leukemia virus). To better understand the molecular pathways leading to cell transformation, we aimed to identify cellular proteins interacting with Tax. METHODS: We used a yeast two-hybrid system to identify interacting cellular proteins. Interactions between Tax and candidate interacting cellular proteins were confirmed by glutathione S-transferase (GST) pulldown assays, co-immunoprecipitation, and confocal microscopy. Functional interactions between Tax and one interacting protein, tristetraprolin (TTP), were assessed by analyzing the expression of tumor necrosis factor-alpha (TNF-alpha), which is regulated by TTP, in mammalian cells (HeLa, D17, HEK 293, and RAW 264.7) transiently transfected with combinations of intact and mutant Tax and TTP. RESULTS: We obtained seven interacting cellular proteins, of which one, TTP, was further characterized. Tax and TTP were found to interact specifically through their respective carboxyl-terminal domains. The proteins colocalized in the cytoplasm in a region surrounding the nucleus of HeLa cells. Furthermore, coexpression of Tax was associated with nuclear accumulation of TTP. TTP is an immediate-early protein that inhibits expression of TNF-alpha at the post-transcriptional level. Expression of Tax reverted this inhibition, both in transient transfection experiments and in stably transfected macrophage cell lines. CONCLUSION: Tax, through its interactions with the TTP repressor, indirectly increases TNF-alpha expression. This observation is of importance for the cell transformation process induced by leukemogenic retroviruses, because TNF-alpha overexpression plays a central role in pathogenesis. [less ▲]Detailed reference viewed: 51 (18 ULg)
Reduced Cell Turnover In Bovine Leukemia Virus-Infected, Persistently Lymphocytotic Cattle
; ; et al
in Journal of Virology (2003), 77(24),Detailed reference viewed: 10 (1 ULg)
Increased cell proliferation-but not reduced cell death-induces lymphocytosis in Bovine Leukaemia Virus-infected sheep
; ; et al
in Abstracts of papers presented at the 2002 meeting of retroviruses (2002, May)Detailed reference viewed: 5 (1 ULg)
Interacting surface of the receptor-binding domain.
GATOT, Jean-Stéphane ; ; et al
in Société Belge de Biochimie et de Biologie moléculaire. (2002, February 22)Detailed reference viewed: 10 (2 ULg)
In vivo cell turnover in BLV-infected sheep.
; ; et al
in Société Belge de Biochimie et de Biologie moléculaire (2002, February 22)Detailed reference viewed: 2 (2 ULg)
Bovine Leukemia Virus Su Protein Interacts With Zinc, And Mutations Within Two Interacting Regions Differentially Affect Viral Fusion And Infectivity In Vivo
GATOT, Jean-Stéphane ; ; et al
in Journal of Virology (2002), 76(16),Detailed reference viewed: 14 (9 ULg)
Oncoviral bovine leukemia virus G4 and human T-cell leukemia virus type 1 p13(II) accessory proteins interact with farnesyl pyrophosphate synthetase
; Vanderplasschen, Alain ; et al
in Journal of Virology (2002), 76(3), 1400-1414
G4 and p13(II) are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13(II) open reading frames ... [more ▼]
G4 and p13(II) are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13(II) open reading frames interferes with viral spread in animal model systems, indicating that the corresponding proteins play a key role in viral replication. In addition, G4 is oncogenic in primary cell cultures and is absolutely required for efficient onset of leukemogenesis in sheep. To gain insight into the function of these proteins, we utilized the yeast two-hybrid system to identify protein partners of G4. Results revealed that G4 interacts with farnesyl pyrophosphate synthetase (FPPS), a protein involved in the mevalonate/squalene pathway and in synthesis of FPP, a substrate required for prenylation of Ras. The specificity of the interaction was verified by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation experiments. Furthermore, confocal microscopy showed that the subcellular localization of G4 was profoundly affected by FPPS. The G4 protein itself was not prenylated, at least in rabbit reticulocyte lysate-based assays. The domain of G4 required for binding to FPPS was restricted to an amphipathic alpha-helix rich in arginine residues. Subtle mutation of this alpha-helix abrogated G4 oncogenic potential in vitro, providing a biological relevance for FPPS-G4 complex formation in cells. Finally, HTLV-1 p13(II) was also found to specifically interact with FPPS (in yeast as well as in GST pull-down assays) and to colocalize with G4 in mitochondria, suggesting a functional analogy between these oncoviral accessory proteins. Identification of FPPS as a molecular partner for p13(II) and G4 accessory proteins opens retrovirus-induced leukemia. [less ▲]Detailed reference viewed: 28 (6 ULg)